Process for increasing RSV surface glycoprotein yields using a mutant strain of RSV

ABSTRACT

A process for producing isolated and purified respiratory syncytial virus (RSV) fusion (F) and attachment (G) glycoproteins in eukaryotic cell cultures infected with RSV cold-passaged, temperature-sensitive mutant subgroup A2 strain cpts-248/404 results in at least a 5-fold increase in F and G protein yields when compared with the parent A2 strain. Immunogenic compositions comprising the F and/or G protein(s) produced by this process can be formulated for in vivo administration to a host to confer protection against disease caused by RSV.

This application is the US national phase of international applicationPCT/US2004/008028 filed on Mar. 17, 2004, which designated the US andclaims priority to US Provisional Application No. 60/455,537, filed Mar.18, 2003. The entire contents of these applications are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a method of identifying a respiratorysyncytial virus (RSV) strain that produces high yields of RSV surfaceglycoproteins. The present invention also relates to the subsequentproduction, isolation and purification of these proteins for their usein immunogenic compositions that are effective in conferring protectionagainst disease caused by RSV.

BACKGROUND OF THE INVENTION

Acute lower respiratory tract disease is the leading contributor tomorbidity and mortality in young children throughout the world.Respiratory syncytial virus (RSV) is the most important viral cause ofserious lower respiratory tract disease in infants and childrenworldwide. It is also a significant cause of lower respiratory tractdisease in the elderly and those with pre-existing chronic cardiac orlung disease such as cystic fibrosis (CF).

The structure and composition of RSV has been elucidated and isdescribed in detail in the textbook “Fields Virology”, ed. by Knipe, D.M. et al., Lippincott Williams & Wilkins, NY (2001), in particular,Chapter 45, pp. 1443-1485, “Respiratory, Syncytial Virus” by Collins,P., Chanock, R. M. and Murphy, B. R.

RSV is an enveloped RNA virus of the family Paramyxoviridae and of thegenus Pneumovirus. The two major protective antigens of RSV are theenvelope fusion (F) and attachment (G) glycoproteins. The F protein issynthesized as a 68 kDa precursor molecule (F₀) which is proteolyticallycleaved into disulfide-linked F₁ (about 48 kDa) and F₂ (about 20 kDa)polypeptide fragments. The unglycosylated G protein (about 33 kDa) isheavily O-glycosylated giving rise to a glycoprotein of apparentmolecular weight of about 90 kDa. Two broad subtypes of RSV have beendefined A and B. The major antigenic differences between these subtypesare found in the G protein while the F protein is more conserved.

Currently, no immunogenic composition to prevent or attenuateRSV-related illness is available. Numerous candidate immunogeniccompositions have been tested over the past thirty years but none havebeen licensed to date. First and second generations of the purifiedfusion protein (designated PFP-1 and PFP-2) immunogenic composition, anRSV subunit immunogenic composition, have been tested in RSVseropositive children and they have been shown to be safe and reasonablyimmunogenic.

The purification of the RSV F and G proteins by immunoprecipitation orpreparative SDS-PAGE provides only small amounts of protein. Thus, thereremains a need for immunogenic compositions effective in conferringprotection against disease caused by RSV, and there is also a need for aprocess that produces high yields of the RSV glycoproteins to meet thedemands of all target populations, such as older children and theelderly.

SUMMARY OF THE INVENTION

The present invention provides a method of identifying an RSV strainthat produces high yields of RSV F and G glycoproteins. This methodcomprises providing a eukaryotic cell culture, infecting the culturewith a live attenuated RSV A2 strain, and determining the glycoproteinconcentration in the infected cell culture, wherein at least a five-foldincrease in F and/or G protein concentration is an indication that theattenuated RSV strain produces high yields of RSV F protein and/or Gprotein when compared with the parent A2 strain. The inventorsdiscovered that the RSV mutant A2 strain cpts-248/404 (ATCC VR 2454)produces more than five times the F protein when grown in, for example,VERO cells at 30° C. than does the parent A2 strain grown at 37° C. SeeTables 7 and 8 below.

Also contemplated is a process for producing purified RSV F and/or Gprotein from eukaryotic cells infected with the RSV mutant straincpts-248/404. This process comprises growing eukaryotic cells infectedwith the RSV mutant strain cpts-248/404 in a culture medium,solubilizing the F and/or G protein(s) from the virus infected cellmembrane, and isolating and purifying the solubilized F and/or Gprotein(s).

The isolation and purification can be effected by standard procedureswell known to those skilled in the art, including chromatography (e.g.,ion exchange, immunoaffinity, and sizing column chromatography),centrifugation, differential solubility, or by any other standardprocedure for the purification of proteins.

Also contemplated is a process for producing an immunogenic compositionfor protecting against disease caused by RSV, wherein said processcomprises growing eukaryotic cells infected with the RSV mutant straincpts-248/404 in a culture medium; solubilizing the F and/or G protein(s)from the virus infected cell membrane; isolating and purifying thesolubilized F and/or G protein(s); and bringing an effective amount ofsaid solubilized F and/or G protein(s) into combination or associationwith a physiologically acceptable carrier.

Also contemplated is the purified RSV F protein produced by this process(designated PFP-3), i.e., in a cell line infected with the RSVcold-passaged, temperature-sensitive mutant subgroup A2 straincpts-248/404, in yields that are at least five times greater than theyields of RSV F protein produced using the parent A2 strain (PFP-2),thereby making the production of RSV F protein (PFP-3) commerciallyfeasible.

Also contemplated is the purified RSV G protein produced by thisprocess, i.e., in a cell line infected with the RSV cold-passaged,temperature-sensitive mutant subgroup A2 strain cpts-248/404, in yieldsthat are at least five times greater than the yields of RSV G proteinproduced using the parent A2 strain, thereby making the production ofRSV G protein commercially feasible.

Also contemplated is the use of an RSV mutant subgroup A2 straincpts-248/404 in the preparation of an RSV envelope fusion (F) proteinand/or RSV attachment (G) glycoprotein.

Also contemplated is an immunogenic composition comprising, in aphysiologically acceptable carrier, an immunoeffective amount of the Fprotein (PFP-3) provided herein. Each immunogenic composition providedherein can be formulated for in vivo administration to a host, which maybe a primate, specifically a human host, to confer protection againstdisease caused by RSV.

Also contemplated is an immunogenic composition comprising, in aphysiologically acceptable carrier, an immunoeffective amount of the Gprotein provided herein. Each immunogenic composition provided hereincan be formulated for in vivo administration to a host, which may be aprimate, specifically a human host, to confer protection against diseasecaused by RSV.

The immunogenic compositions of the invention can be formulated asmicroparticles, capsules, ISCOMs or liposomes. The immunogeniccompositions can further comprise at least one other immunogenic orimmunostimulating material, which may be at least one adjuvant or atleast one immunomodulator.

The immunogenic compositions provided herein can be formulated tocomprise at least one additional immunogen, which conveniently maycomprise the other RSV protein (i.e., G in addition to F or F inaddition to G), or a human Parainfluenza virus (PIV) protein from PIV-1,PIV-2 and/or PIV-3 such as F and HN proteins. However, other immunogens,such as from Chlamydia, polio, hepatitis B, diphtheria toxoid, tetanustoxoid, influenza, haemophilus, B. pertussis, pneumococci, mycobacteria,hepatitis A and Moraxella also can be incorporated into thecompositions, as polyvalent (combination) immunogenic compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows rate nephelometry titration of PFP-2 and PFP-3 when using amixture of polyclonal antibodies made against PFP-2.

FIG. 2 shows the capacity of PFP-2 and PFP-3 to generate cell-mediatedimmune responses in BALB/c mice. BALB/c mice were immunized(intramuscularly) on weeks 0 and 4 with 3 μg F protein from eithermutant 248/404 (circles) or A2 (triangles) strains of RSV. Theimmunogenic compositions were admixed with QS-21 (20 μg/dose). Controlmice were immunized by experimental infection with the A2 strain of RSV(filled squares) or received intranasally an equal volume of mockinfected HEp-2 cell lysate (open squares). Two weeks after the lastimmunization the mice were challenged with the A2 strain of RSV.Pulmonary inflammatory cells were isolated by bronchoalveolar lavagefive days later and tested directly ex vivo against syngeneic infected(RSV A2, solid lines) or control (dashed lines) targets. There were fivemice per group.

DETAILED DESCRIPTION OF THE INVENTION

Up to now, the parent virus strain (A2) has been the source of F protein(PFP-2) for an RSV subunit immunogenic composition. Concerns have beenraised, however, regarding the ability to produce quantities of native Fprotein from the A2 strain of RSV sufficient to meet the demands of alltarget populations. Based on F protein yields from the parent strain A2,development of a manufacturing-scale process for F protein was notfeasible. Surprisingly, F protein production using the RSVtemperature-sensitive mutant strain cpts-248/404 resulted in a greaterthan five-fold increase in F protein (PFP-3) yields as compared with theA2 strain, which made development of a manufacturing-scale process for Fprotein feasible. Since the use of cpts-248/404 makes F proteinproduction on a large scale more feasible, preclinical efforts focusedon demonstrating immunological and biochemical equivalence of F proteinspurified from the A2 and mutant cpts-248/404 strains of RSV (see below).

As discussed above, the present invention provides the biochemicallypurified F protein from the cold-passaged, temperature-sensitive mutantstrain cpts-248/404 (PFP-3). This is a cold-passaged strain of RSV A2(cp-248) that was further attenuated by chemical mutagenesis(5-fluorouracil) in VERO cells. This attenuated strain was found to be1000-fold restricted in replication compared to wild-type and 100-foldrestricted in replication compared to the parent A2 strain at 37° C. TheRSV mutant strain cpts-248/404 has been shown to produce greater thanfive times the F protein levels during infection of VERO cells onmicrocarriers in a bioreactor at 30° C. when compared with the parent A2strain grown at 37° C.

The cpts-248/404 virus is grown in any cell line that supports RSVgrowth, such as, for example, VERO, MRC-5, FRhL, CEF (chicken embryofibroblast) and PER.C6 cells. The infection process proceeds for abouteight days until there is greater than 90% syncytial formation andcellular detachment from the microcarriers. At that time, the infectedcell culture is lysed in situ with 0.5% v/v Triton X-100 for 2 hours at30° C. The lysed cell culture supernatant is clarified by depthfiltration followed by ultrafiltration/diafiltration. The diafilteredlysate is applied to an anion exchange column and the flow throughfraction, containing the F protein, is collected. The flow throughfraction is diafiltered utilizing a spiral wound ultrafiltration moduleand applied to a cation exchange column. The F protein is eluted with astep gradient of higher ionic strength. The cation exchange pool isdiafiltered utilizing an ultrafiltration module and applied to ahydroxyapatite column. The F protein is eluted with a step gradient ofhigher phosphate concentration and higher pH. Fractions containing Fprotein are pooled, terminally sterile-filtered, and frozen at −70° C.until formulation.

The current process to purify G protein from cpts-248/404-infected Verocells starts with the same process as for the F protein purification(PFP-3). Identical to the process for F protein, the infection processproceeds for eight days until there is greater than 90% syncytialformation and cellular detachment from the microcarriers. At that time,the infected cell culture is lysed in situ with 0.5% v/v Triton X-100for 2 hours at 30° C. The lysed cell culture supernatant is clarified bydepth filtration followed by ultrafiltration/diafiltration. Thediafiltered lysate is applied to an anion exchange column and the flowthrough fraction, containing the F protein, is collected. The G proteinis retained by the DEAE column and eluted with a step elution to 200-mMNaCl.

The G protein from the DEAE pool is then applied to a boronic acidaffinity chromatography column with the boronate ligand3-aminophenylboronic acid (APBA) utilizing a 9-atom spacer at pH 8.5.The esterification of boronate with 1,2-cis-diol provides the basis forthe purification of macromolecules that possess two hydroxyl groups onadjacent carbon atoms. The G protein is eluted from the column with theaddition of 500 mM Tris buffer. The APBA pool containing G protein isthen dialyzed and applied to a hydroxyapatite column (Type II, 80 μm)and eluted with an increase in potassium phosphate concentration. Thehydroxyapatite column pool containing G-protein is dialyzed and appliedto a lectin (wheat germ) affinity chromatography column and eluted with300 mM N-acetyl-glucosamine.

Various immunological and biochemical assays were employed to comparethe F proteins from the mutant virus strain (cpts-248/404, PFP-3) andthe parent strain (A2, PFP-2). From an immunological standpoint, nostatistically significant differences were observed. The antigenicity ofthe two proteins was determined by both rate nephelometry andcompetition ELISAs using monoclonal antibodies and human convalescentsera. Rate nephelometry, which quantifies the rate of change inmolecular size as an antigen/antibody complex forms, was similar betweenthe two proteins when using a mixture of polyclonal antibodies madeagainst PFP-2 (FIG. 1). The conservation of B cell epitopes between thetwo proteins was evaluated by competitive ELISA. In those studies, theability of PFP-2 and PFP-3 to inhibit the binding of seven differentmonoclonal antibodies or a mixture of polyclonal antibodies to eitherthe homologous or heterologous protein adsorbed to the ELISA plate(Tables 1 and 2) was compared. It should be noted that all thesemonoclonal antibodies have previously been shown to neutralize virusinfectivity. It was found that PFP-3 caused greater than 95% inhibitionof the monoclonal antibodies to PFP-2 and PFP-3. Similarly, PFP-2caused >80% inhibition and in most cases >93% inhibition of themonoclonal antibodies to PFP-3 and PFP-2. This is strong evidence thatthese major B cell epitopes, which represent regions of the moleculethat can stimulate protective antibody responses, are conserved betweenthe two proteins, even though there are minor differences in theirsecondary structures (see below).

The immunogenicity and efficacy of the two F proteins in a BALB/c mousemodel were evaluated. The data (Table 3) suggest that the systemichumoral immune responses induced by either protein when adsorbed toAl(OH)₃ are equivalent. Moreover, the immunity engendered by eitherprotein protects the lower respiratory tract of BALB/c mice againstexperimental infection with RSV to the same degree.

To evaluate the ability of the two F proteins to induce cell-mediatedimmune responses, mice were immunized with protein formulated withSTIMULON™ QS-21 adjuvant (Antigenics, Framingham, Mass.) (QS-21) and theinduction of virus-specific killer cells was measured by an in vitrocytolytic assay. The data indicate that the systemic cell-mediatedimmune responses generated after immunization with either PFP-2 or PFP-3were equivalent (FIG. 2). The data with QS-21 further show that the twoproteins were able to generate equivalent systemic humoral immuneresponses (Table 4) along with equivalent complement-assisted andcomplement-independent neutralizing antibody titers in a two-doseimmunization schedule (Table 4).

Various biochemical assays were employed to compare the primary,secondary, tertiary, and quaternary structures of the two proteins alongwith any differences in their glycosylation patterns. As might beexpected from chemical mutagenesis and subsequent attenuation, PFP-3displays four amino acid substitutions in its primary sequence, two inthe F₁ and two in the F₂ polypeptides. Two changes result in a chargechange, one change is to proline, and the other change is veryconservative (Table 5). Circular dichroism analysis indicates that PFP-3contains more beta-sheet structure than does PFP-2 (data not shown). Theaggregation state of the two purified bulks, as measured by analyticalultracentrifugation and multi-angle laser light scattering (MALLS)appeared to be similar, with the proteins existing as aggregates >500 kDin molecular weight (data not shown). Various SDS-PAGE conditions, i.e.heated vs. nonheated, and reduced vs. nonreduced, displayed similarbanding patterns between PFP-3 and PFP-2 of dimeric, trimeric, andoligomeric structure (data not shown), all of which have been implicatedin the usefulness of the protein as an immunogenic composition. PFP-3 F₁and F₂ polypeptides both display smaller apparent molecular weights thantheir PFP-2 counterparts by SDS-PAGE analysis. Carbohydrate analysis,utilizing fluorescence-activated carbohydrate electrophoresis (FACE),reveals subtle differences in the sialic acid, mannose, and fucosecontent of the N-linked carbohydrates (data not shown).

In summary, it has been shown that even though subtle physico-chemicaldifferences between the two molecules were found to exist, PFP-2 andPFP-3 are antigenically and immunologically equivalent.

TABLE 1 A2 vs. 248/404 - Monoclonal Competitive ELISA Percent InhibitionA2 Binding Assay 248/404 Binding Assay Monoclonal A2 248/404 A2 248/404Antibody (Homo) (Hetero) (Hetero) (Homo) L4 97.5 94.6 98.8 97.3 A5 98.493.8 >99 99.0 133-1H 98.1 93.1 >99 96.2 143-6C 98.4 95.0 >99 98.11129 >99 94.7 >99 95.9 1269 96.6 82.3 >99 >99 1243 >99 94.4 >99 98.9

TABLE 2 A2 vs. 248/404 - Polyclonal Competitive ELISA Percent InhibitionA2 Binding Assay 248/404 Binding Assay A2 248/404 A2 248/404 Sera (Homo)(Hetero) (Hetero) (Homo) C587645 (pooled human) 95.5 93.9 77.9 87.0C587769 (pooled human) 96.8 92.5 75.2 85.6 Control C (individual) 94.093.4 85.4 80.7 Control D (individual) 97.1 94.6 77.1 83.8

TABLE 3 A2 vs. 248/404 The systemic humoral and protective immuneresponses of BALB/c mice 4 weeks after primary immunization with PFP-2and PFP-3 (T97-0156) MEAN ANTIBODY TITERS¹ F Protein Anti-F Protein(×1000) Neutralizing GMT RSV (Dose) IgG IgG1 IgG2a +C′ −C′ (log₁₀)²(248/404) 145.1^(a) 86.9^(a) 1.4 20 <20 2.1 ± 1.0 (3000 ng) (248/404) 52.0^(b) 18.8^(a) <0.1 <20 <20 2.5 ± 1.2 (300 ng) (248/404)  14.5^(c)10.7^(c) <0.1 <20 <20 3.7 ± 0.5 (30 ng) A2 122.3^(d) 75.8^(d) 1.1 <20<20 2.8 ± 0.9 (3000 ng) A2  66.0^(c) 28.6^(e) <0.1 <20 <20 2.6 ± 1.2(300 ng) A2  27.9 15.1 <0.1 <20 <20 2.6 ± 1.1 (30 ng) RSV (A2) 159.3 8.9 26.5 78 <20 <1.4 ± 0.1  PBS  <1.0 <0.1 <0.1 <20 <20 4.4 ± 1.0¹BALB/c mice were primed with the indicated doses of fusion (F) proteinfrom either the A2 or 248/404 strains of RSV. The proteins were adsorbedto aluminum hydroxide (AlOH) adjuvant. Control mice were injected withAlOH in PBS alone, or were infected with the A2 strain of RSV.Four weeksafter primary vaccination sera were collected for the determination ofgeometric mean endpoint anti-F protein total and subclass IgG antibodytiters by ELISA. Neutralizing antibody titers were also revealed by theplaque reduction neutralization test against A2 strain of virus in thepresence (+)and absence (−) of 5% complement. There were 5 mice pergroup. ²The geometric mean titers (GMT) of RSV are expressed per gram ofpulmonary tissue. The titers were determined 4 days after challenge withthe A2 strain of RSV. ^(a)P < 0.05 when compared to sera from micevaccinated with either 300 or 30 ng F protein from strain 248/404. P >0.05 when compared to sera from mice vaccinated with 3000 ng F proteinfrom strain A2. ^(b)P < 0.05 when compared to sera from mice vaccinatedwith 30 ng F protein from strain 248/404. P > 0.05 when compared to serafrom mice vaccinated with 300 ng F protein from strain A2. ^(c)P < 0.05when compared to sera from mice vaccinated with 30 ng F protein A2strain. ^(d)P < 0.05 when compared to sera from mice vaccinated witheither 300 or 30 ng F protein strain A2. ^(e)P < 0.05 when compared tosera from mice vaccinated with either 30 ng F protein from strain248/404 or 300 ng F protein from strain A2 ^(f)P > 0.05 when compared tosera from mice vaccinated with 30 ng F protein from strain A2.

TABLE 4 The systemic humoral immune responses of BALB/c mice 2 and 4weeks after primary and secondary immunization with PFP-2 and PFP-3(T97-2502). GEOMETRIC MEAN ANTIBODY TITERS (Log₁₀)² Anti-F ProteinNeutralizing PRIMARY IgG IgG1 IgG2a +C′ −C′ F Protein (248/404) 5.7 ±0.1 4.9 ± 0.1 4.9 ± 0.1 2.5 ± 0.4 <1.3 F Protein (A2)  6.1 ± 0.1^(a) 5.3 ± 0.1^(a)  5.3 ± 0.1^(a)  2.7 ± 0.4^(b) 1.4 ± 0.2 RSV (A2) 4.8 ±0.1 3.6 ± 0.3 4.1 ± 0.1 2.1 ± 0.3 <1.3 PBS (QS-21) <1.7 <1.7 <1.7 <1.3<1.3 SECONDARY IgC IgG1 IgG2a +C² −C² F Protein (248/404) 7.1 ± 0.1 6.2± 0.1 6.1 ± 0.1 3.6 ± 0.2 2.7 ± 0.2 F Protein (A2)  7.0 ± 0.1^(b)  6.1 ±0.1^(b)  6.0 ± 0.1^(b)  3.7 ± 0.2^(b)  3.0 ± 0.3^(b) RSV (A2) 5.7 ± 0.44.3 ± 0.3 5.2 ± 0.1 3.3 ± 0.4 2.6 ± 0.5 PBS (QS-21) <1.7 <1.7 <1.7 <1.3<1.3 ¹BALB/c mice were vaccinated on weeks 0 and 4 with 3 μg F proteinfrom either the 248/404 or A2 strains of RSV. The F protein wasformulated with 20 μg QS-21. There were 5 mice per group. ²The geometricmean antibody titers were determined on sera collected 2 and 4 weeksafter primary and secondary vaccination respectively. Endpoint IgG andneutralizing antibody titers were determined by ELISAand the plaquereduction neutralization test respectively.The latter antibody titerswere determined in the presence (+) and absence (−) of 5% complement(C¹). ^(a)P < 0.05 when compared to sera from mice vaccinated with FProtein from strain 248/404. ^(b)P > 0.05 when compared to sera frommice vaccinated with F Protein from strain 248/404.

TABLE 5 A2 vs. 248/404 Sequence Differences in F-Protein NucleotideAmino Acid cpts- Charge Position Position A2 (M6) 248/404 Change 5857 66Lys (K) Glu (E) −2 5962 101 Gln (Q) Pro (P) 0 6313 218 Glu (E) Ala (A)+1 7228 523 Thr (T) Ile (I) 0Preparation and Use of the Immunogenic Composition

Immunogenic compositions may be prepared from the F protein (PFP-3) ofRSV as disclosed herein. The immunogenic composition elicits an immuneresponse that produces antibodies, including anti-RSV and anti-Fantibodies. Such antibodies may be viral neutralizing and/or anti-fusionantibodies.

Immunogenic compositions may be prepared as injectables, as liquidsolutions, suspensions or emulsions. The active immunogenic ingredientor ingredients may be mixed with pharmaceutically acceptable excipientsthat are compatible therewith. Such excipients may include water,saline, dextrose, glycerol, ethanol, and combinations thereof. Theimmunogenic compositions may further contain auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness thereof. The immunogenic compositions may beadministered parenterally, by injection subcutaneously, intradermally orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. Alternatively, othermodes of administration including suppositories and oral formulationsmay be desirable. For suppositories, binders and carriers may include,for example, polyalkalene glycols or triglycerides. Such suppositoriesmay be formed from mixtures containing the active immunogenic ingredient(s) in the range of about 0.5 to about 10%, such as about 1 to 2%. Oralformulations may include normally employed carriers, such aspharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and containabout 1 to 95% of the active ingredient(s), such as about 20 to about75%. The immunogenic preparations are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, immunogenic and protective. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies, and if needed, to produce a cell-mediated immune response.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges arereadily determinable by one skilled in the art. Suitable regimes forinitial administration and booster doses are also variable, but mayinclude an initial administration followed by subsequent boosteradministrations. The dosage may also depend or the route ofadministration and will vary according to the size of the host.

The concentration of the active ingredient protein in an immunogeniccomposition according to the invention is in general about 1 to 95%. Animmunogenic composition that contains antigenic material of only onepathogen is a monovalent composition. Immunogenic compositions thatcontain antigenic material of several pathogens are polyvalent(combination) compositions and also within the scope of the presentinvention. Such polyvalent compositions contain, for example, materialfrom various pathogens (such as PIV-1, PIV-2 and/or PIV-3) or fromvarious strains of the same pathogen, or from combinations of variouspathogens.

In certain embodiments, the immunogenic composition will comprise one ormore adjuvants. As defined herein, an “adjuvant” is a substance thatserves to enhance the immunogenicity of an immunogenic composition ofthis invention. Thus, adjuvants are often given to boost the immuneresponse and are well known to the skilled artisan.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as, for example,

-   -   (a) MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene,        0.5% Tween 80, and 0.5% Span 85 (optionally containing various        amounts of MTP-PE (see below, although not required)) formulated        into submicron particles using a microfluidizer such as Model        110Y microfluidizer (Microfluidics, Newton, Mass.),    -   (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5%        pluronic-blocked polymer L121, and thr-MDP (see below) either        microfluidized into a submicron emulsion or vortexed to generate        a larger particle size emulsion, and    -   (c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, Mont.)        containing 2% Squalene, 0.2% Tween 80, and one or more bacterial        cell wall components from the group consisting of 3-O-deaylated        monophosphorylipid A (MPL™) described in U.S. Pat. No. 4,912,094        (Corixa), trehalose dimycolate (TDM), and cell wall skeleton        (CWS), preferably MPL+CWS (Detox™);

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (U.S. Pat. No. 5,057,540) may be used or particlesgenerated therefrom such as ISCOMs (immunostimulating complexes);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside, which is alsoknow as 529 (formerly known as RC529), which is formulated as an aqueousform or as a stable emulsion, synthetic polynucleotides such asoligonucleotides containing CpG motif(s) (U.S. Pat. No. 6,207,646);

(5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte magrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor nucrosis factor (TNF), etc.;

(6) detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT) either in a wild-type or mutant form, for example,where the glutamic acid at amino acid position 29 is replaced by anotheramino acid, preferably a histidine, in accordance with publishedinternational patent application number WO 00/18434 (see also WO02/098368 and WO 02/098369), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129(see, e.g., WO 93/13302 and WO 92/19265); and

(7) other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

RSV subunit immunogenic compositions will provide a strategy forimmunization of high-risk children, adults, and the elderly againstRSV-induced lower respiratory tract disease.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

Dulbecco's Modification of Eagles Medium (DMEM) and fetal bovine serum(FBS), with or without Pluronic F68, culture medium was used for cellculture and virus growth. The cells used in these Examples werequalified African green monkey kidney cells (VERO) obtained from Wyeth'sMaster Cell Bank. The RS virus used was the RSV mutant subgroup A2strain cpts-248/404 (ATCC VR 2454), which was obtained from the NationalInstitute of Allergy and Infectious Diseases (NIAID), Bethesda, Md.,where it was developed (U.S. Pat. No. 5,922,326, incorporated herein byreference).

Example 1

This Example illustrates the screening method utilized to identifyattenuated strains of RSV capable of producing high yields of RSV fusion(F) protein in VERO cell microcarrier cultures.

A VERO cell culture was initiated from frozen ampoules of Wyeth'squalified Working Cell Bank. The ampoules were thawed and inoculatedinto cell factories (Nunc) for the initial stage. The cells were grownat 37° C.±0.5° C. without CO₂. The media used for cell cultivation wasDMEM supplemented with 10% (v/v) FBS. Two 100% media exchanges wereperformed for optimal cell growth. Upon confluency, cells were releasedfrom the surface by treatment with trypsin and inoculated onto Cytodex 1microcarriers in a 3.6-L (working volume) bioreactor equipped with anenvironmental control. The cells were cultivated in the bioreactor undercontrolled conditions until greater than 90% confluency (3-4×10⁶cells/mL) was reached. The cells were infected with an attenuated RSV A2strain at a multiplicity of infection (MOI) of 0.001. Harvest wasperformed eight days post-infection, when greater than 90% syncytialformation and cell detachment from the microcarriers were achieved.Harvest samples containing microcarriers were frozen at −70° C. foranalysis of RSV F protein concentration using an F protein specificmonoclonal antibody (L4) in a capture ELISA format. Frozen harvestretention samples were thawed at room temperature and 25 μL of 10%Triton X-100 non-ionic detergent was added to 500 μL of thawed sample inan Eppendorf tube for solubilization of the F protein. Samples wereplaced on a rotating rocker at slow speed for 1 hour at 15-30° C. Themicrocarriers were allowed to settle and the supernatant was transferredto an L4-coated 96-well microtiter plate containing an F proteinstandard. After a one-hour incubation at room temperature, the wellswere washed and probed with a primary antibody (Rabbit anti-F-protein).After a one-hour incubation at room temperature, the wells were washedand probed with a secondary antibody (Goat anti-Rabbit HRP). After aone-hour incubation at room temperature, the wells were washed andprobed with Horseradish Peroxidase Enzyme Substrate (ABTS). Colordevelopment proceeded for 30 minutes and plates were read at awavelength of 405 nm. A standard curve was fitted to a log-cubicequation using the optical density (OD) values from the F proteinstandard. The concentration of F protein from the unknown attenuated RSVstrain was determined by averaging all consecutive OD values thatcoincide within the range of OD values used for the standard curve. Bythis method, strains of RSV capable of producing at least a five-foldincrease in RSV F protein yields as compared with the parent A2 strainwere identified. See Tables 6 and 7 below.

TABLE 6 RSV Strains Screened for Increased F Protein Levels AverageF-Protein Level* RSV Strain (μg/mL) A2 (parent) (PFP-2) 5.0 cpts248/404(PFP-3) 22.0 RSS-2 6.5-11 Cp52 (B-strain) 0.57 RA2cp248/404 13.2rA2cp248/404/ΔSH 7.6 rA2cp248/404/1030 20.1 rA2cp248/404/1030/ΔSH 4.1rA2cpΔNS2 1.1 rA2cp248/404/ΔNS2 1.5 rA2cp530/1009/ΔNS2 1.7rA2cp530/1009/404 1.0 rABcp248/404/1030 1.0 rABcp248/404/ΔSH 1.8RABcp530/1009/ΔNS2 0.6 rABcp248/404/ΔNS2 1.9 rABcpΔNS2 0.5 ts 1c9097C-99 9.5 *Value is average from preseed production, master virusseed, and/or clinical production.

Example 2

This Example illustrates the production of RSV on a mammalian cell lineon microcarriers in a 30-L controlled bioreactor.

A VERO cell culture was initiated from frozen ampoules of Wyeth'squalified Working Cell Bank (BB-MF 6735) at passage 134. The ampouleswere thawed and inoculated into 6000 cm² cell factories (Nunc) for theinitial stage at a cell density of 2±0.5×10⁴ cells/cm². The cells weregrown at 37° C.±0.5° C. without CO₂. The media used for cell cultivationwas DMEM supplemented with 10% (v/v) FBS. Two 100% media exchanges wereperformed for optimal cell growth. Upon confluency, cells were releasedfrom the surface by treatment with trypsin and inoculated onto Cytodex 1microcarriers at 0.5-0.8×10⁶ cells/mL in a 3.6-L (working volume)bioreactor equipped with an environmental control. The cells werecultivated in the bioreactor under controlled conditions until greaterthan 90% confluency (3-4×10⁶ cells/mL) was reached. Cells were thentrypsinized off the microcarriers and scaled up into a 23-L (workingvolume) bioreactor. VERO cells were cultivated in the bioreactor undercontrolled conditions until greater than 90% confluency (3-4×10⁶ c/mL)was reached. The media used for initial cell cultivation in bothbioreactors was DMEM with 5% (v/v) FBS. Following a one- to two-dayperiod of batch-wise cell growth, the culture was perfused with DMEMwith 1% (v/v) FBS for three to six culture volume exchanges. Thefollowing parameters were monitored and/or controlled cell growth,glucose (>1 g/L) and glutamine (2-4 mM), dissolved oxygen (50%), pH(7.4) and temperature (37° C.). At 90% confluency the bioreactortemperature was reduced to 30±0.5° C. and perfusion was turned off whileall other environmental conditions were maintained. When the temperatureequilibrated to 30±0.5° C., cells were infected with the qualified RSVsubgroup A2 strain cpts-248/404 virus seed at a multiplicity ofinfection (MOI) of 0.001. Perfusion remained off for 24±5 hours to allowvirus adsorption and a batch addition of glucose and glutamine was made(to reach 3 g/L glucose and 4 mM glutamine). Perfusion was thencontinued during infection for up to 4 days. Harvest was performed eightdays post-infection, when greater than 90% syncytial formation and celldetachment from the microcarriers were achieved. Further processing ofthe harvested material is described in Example 3.

Example 3

This Example illustrates the process of purifying subunit RSV PFP-3 froma viral concentrate,

Sterile lysis buffer [10% (v/v) Triton-X-100 in PBS, pH 7.4±0.1] wasadded to the bioreactor to a final detergent concentration of 0.5%(v/v). The culture was agitated during the lysis period of 2-3 hours at30±0.5° C. At the end of lysis in situ, the microcarriers were allowedto settle and the lysed culture supernatant was clarified through aSeitz® 700 (6-15 μm) cellulose depth filter (0.3 ft²/L harvest) followedby a Seitz® 100 (1-3 μm) cellulose depth filter (0.3 ft²/L harvest). Theclarified supernatant was aseptically transferred to a holding vesselfor storage at 2-8° C. prior to purification.

The clarified viral harvest was concentrated five- to 10-fold usingeither spiral wound or plate-and-frame ultrafiltration (0.5 ft²/Loriginal harvest) and diafiltered with about 10 volumes of 75 mMTris-Cl, 0.1% Triton X-100, pH 8 (>100 L) utilizing a 30K Amicon spiralwound, regenerated cellulose or PALL Centrasette Omega® polyethersulfonemodule. The diafiltered lysate was applied to an anion exchange column(DEAE Sepharose or MacroPrep) equilibrated with 75 mM Tris-Cl, 0.1%Triton X-100, pH 8, and the flow through fraction containing theF-protein was collected. The flow-through fraction was concentratedabout two-fold using either spiral wound or plate-and-frameultrafiltration (0.3 ft²/L original harvest) and diafiltered with about10 volumes of 20 mM sodium acetate, 100 mM NaCl, 0.1% Triton X-100, pH 4(approximately 70 L) utilizing either a 30K Millipore spiral woundPrepScale®, polyethersulfone or PALL Centrasette Omega® polyethersulfonemodule, and then applied to a cation exchange column (CM Sepharose FF).The F protein was eluted with a step gradient to 350 mM NaCl.

The cation exchange pool was concentrated about two-fold using eitherspiral wound or plate-and-frame ultrafiltration (0.05 ft²/L originalharvest) and diafiltered with about 10 volumes of 10 mM KPO₄, pH 6(approximately 25 L) utilizing either a 30K Millipore spiral woundPrepScale®, polyethersulfone or PALL Centramate Omega® polyethersulfonemodule, and applied to a hydroxyapatite column (Type II, 80 μM)equilibrated with 10 mM KPO₄, 0.1% Triton X-100, pH 6. The F protein waseluted with a step gradient to 150 mM KPO₄, 0.1% Triton X-100, pH 7.Fractions containing F protein were pooled, terminally sterile-filtered,and frozen at −70° C. until formulation.

The F protein bulk was analyzed by SDS-PAGE and found to be ≧90% pure.

TABLE 7 Comparison of PFP-3 purification process utilizing cpts-248/404or the parent A2 strain. A2 cpts-248/404 Multiplicity of Infection (MOI)0.001 0.001 F-Protein @ Harvest (μg/mL) 5.3 22.4 Virus Titer (pfu/mL) @Harvest 4 × 10⁷ 2 × 10⁸ Cell Concentration (cells/mL) @ Infection 3 ×10⁶ 3 × 10⁶ F Protein Recovery (%) 25 25 F-Protein Recovery (mg/Lharvest) 1.0 5.6 50 μg Doses/L harvest 20 110

Example 4

This Example illustrates the immunogenicity of a PFP-3 immunogeniccomposition in RSV seropositive children with cystic fibrosis.

In a phase II, multi-center, adjuvant-controlled trial, 151 childrenreceived the adjuvant control (aluminum phosphate) and 143 received thePFP-3 immunogenic composition. The F protein accounted for over 95% ofthe total viral protein content in the immunogenic composition and the Gprotein for less than 2%. Children randomized to receive the PFP-3immunogenic composition received a 0.5 ml dose containing 30 μg of PFP-3and 0.5 mg of aluminum phosphate (0.125 mg dose of aluminum), whilechildren in the adjuvant-control group received a 0.5 ml dose containing0.5 mg of aluminum phosphate (0.125 mg of aluminum). The immunogeniccompositions were administered intramuscularly in the deltoid or lateralthigh. Blood samples were obtained at pre-immunization, 28 dayspost-immunization and end-of-study and assayed for neutralizingantibodies (Nt Ab) and binding antibodies (Bd Ab) to the F protein.

Tests for Nt Ab to RSV subgroups A (RSV/A) and B (PSV/B) were performedby a microneutralization assay in 96 well microtiter plates with HEp-2cells as previously described (Piedra P. A., Glezen W. P., Kasel J. A.,et al. “Immunogenic composition 1995; 13:1095-1101, 1995; Groothuis J.R., King S. J., Hogerman D. A., et al.” J. Infect. Dis., 177:467-469,1998). An enzyme-linked immunosorbent assay (ELISA) was used to measureserum IgG binding antibodies (Bd Ab) to the F (ELISA-F) and G (ELISA-G)proteins as previously described. A rise in Ab titer by four folds orgreater is considered a significant antibody response. Comparing thepercentage of children in the adjuvant-control and PFP-3 immunogeniccomposition groups who experienced a four-fold or greater rise in Abtiter at 28 days post-immunization, it was found that nearly all (97%)had a four-fold or greater ELISA-F Bd Ab rise compared to 1 % of theadjuvant-control cohort. A significant Nt Ab rise to RSV/A and RSV/B wasobserved in 67% and 55% of the PFP-3 immunized children compared to 2%and 3%, respectively, of the adjuvant-control cohort (Table 8 below).

TABLE 8 Immunogenicity of the PFP-3 immunogenic composition AdjuvantTest Interval control PFP-3 P Value RSV/A (GMT log 2) Pre-immunization 5.5 ± 1.5* 5.4 ± 1.6 0.49 28 days post-immunization 5.5 ± 1.6 7.9 ± 1.8<0.001 RSV/A (≧4-fold rise) 28 days post-immunization 3/150 (2%) 96/143(67%) <0.001 RSV/B (GMT log 2) Pre-immunization 6.5 ± 2.3 6.4 ± 2.4 0.8228 days post-immunization 6.6 ± 2.3 8.4 ± 1.9 <0.001 RSV/B (≧4-foldrise) 28 days post-immunization 4/149 (3%) 78/143 (55%) <0.001 ELISA-F(GMT log 2) Pre-immunization 13.2 ± 1.6  13.1 ± 1.7  0.49 28 dayspost-immunization 13.1 ± 1.7  17.9 ± 1.2  <0.001 ELISA-F (≧4-fold rise)28 days post-immunization 2/149 (1%) 139/143 (97%)  <0.001 ELISA-G (GMTlog 2) Pre-immunization 9.0 ± 1.9 9.1 ± 1.6 0.58 28 dayspost-immunization 8.8 ± 1.9 9.3 ± 1.6 0.01 ELISA-G (≧4-fold rise) 28days post-immunization 1/149 (1%) 4/143 (3%) 0.21 *serum geometric meanantibody titer in log 2 and standard deviation RSV/A = neutralizingantibody to RSV/A; RSV/B = neutralizing antibody to RSV/B; ELISA-F =binding antibody to F protein; ELISA-G = binding antibody to G protein.Comparisons between categorical variables were conducted using theChi-square test or Fisher's exact test for expected cell counts lessthan 5, and the two-sample t test was used for continuous variables.

1. A method of identifying an attenuated respiratory syncytial virus (RSV) strain that produces high yields of RSV surface glycoprotein F when compared with the parent A2 strain, which method comprises: providing a eukaryotic cell culture; infecting the eukaryotic cell culture with a live, attenuated RSV strain at 30° C.; harvesting the infected cell culture; solubilizing the F protein in the harvested culture; and subsequently determining the glycoprotein F concentration in the harvested culture, wherein at least a five-fold increase in glycoprotein F concentration produced when the attenuated RSV strain is grown in the cell culture at 30° C. is an indication that the attenuated RSV strain produces high yields of RSV F glycoprotein when compared with the parent A2 strain grown at 37° C.
 2. The method of claim 1, wherein the identified attenuated RSV strain is the RSV mutant strain cpts-248/404.
 3. The method of claim 1, wherein the eukaryotic cell culture is a VERO, MRC-5, FRhL, CEF or PER.C6 cell culture.
 4. The method of claim 1, wherein the solubilizing comprises adding detergent to the harvested culture. 